Project Summary The neuroepigenetic model of learning and memory posits that a number of critical epigenetic changes in the active neuron regulate its firing properties in an acute or chronic manner and thereby provide the cell with a form of molecular memory. Driven in part by salient sensory experiences, these changes can potentially modulate behavior for the entire remaining lifespan of the animal. Because drug addiction is a state of chronic maladaptation dependent on broad molecular and physiological plasticity, the neuroepigenetic hypothesis of addiction is an emerging area of research. Psychostimulant reward in rodents has been shown to modulate the neuronal chromatin state. Dynamic DNA methylation in the nucleus accumbens, a central hub of reward processing that integrates drug-induced dopaminergic neurotransmission from the midbrain, was also shown to regulate cocaine sensitization and contextual reward memory. However, little is known about the role or mechanism of demethylation in the striatum. Recent evidence points to a sequential mechanism of demethylation involving oxidation of 5-methylcytosine and subsequent base-excision and repair to the default unmethylated base. This proposal will examine the breadth and functionality of DNA demethylation in the nucleus accumbens in reward learning and gene expression. A dissociated primary culture of striatal neurons will be used to examine the effect of dopamine on transcription and associated DNA methylation. Additionally, multiplex transcriptional control with CRISPR technologies will be used for global regulation of demethylation factors to determine the role of demethylation on learning-related gene expression and cocaine reward behavior. Finally, because the role of site-specific epigenetic dynamics in the brain is poorly understood, this study will utilize a novel fusion construct to direct single-locus demethylation in the striatum. The results of this proposal will expand the current understanding of molecular mechanisms by which drugs of abuse hijack the reward system and, in the long term, will offer novel insights into the potential effectiveness of epigenetic manipulation in addiction therapy.